RNA-Seq analyses of leaf samples collected before and 24 h after P. viticola inoculation from T39-treated and control plants resulted in the identification of 7,024 differentially expressed genes. These genes formed 10 clusters of different expression profiles, highlighting the complex transcriptional reprogramming of T39-induced resistance. T39 treatment directly affected the expression of grapevine genes and to a greater extent enhanced grapevine response to P. viticola inoculation, indicating a dual effect of T39. At the sampling time points selected in this analysis, we showed that a limited number of changes in gene expression were caused by T39 treatment and that more intense transcriptional reprogramming took place after pathogen inoculation. In particular, opposing modulation of genes related to response to stress and protein metabolism was observed in T39-treated and control plants at 24 h after P. viticola inoculation, indicating inhibition of disease-related processes and induction of active defence in T39-treated grapevines. Based on the expression profiles, genes directly modulated by T39, as well as genes with reinforced or specific modulation in T39-treated plants after pathogen inoculation are strong candidates for activation of plant self-protection and consequent inhibition of disease-related processes and symptoms development.
Grapevine processes directly affected by Trichoderma harzianum T39
Analysis of the expression profiles revealed a set of genes directly modulated by T39 (182 up- and 161 down-regulated) and whose expression was not affected by the subsequent pathogen inoculation (cluster 1). Several genes of cluster 1 were involved in signal transduction processes, indicating that they may be related to the initial events of recognition of the beneficial microorganism and induction of resistance, as recently demonstrated by the proteomic analysis of T39-induced resistance
. Enzymes known to mediate microbial recognition and to trigger defence responses in plant species
 were up-regulated by T39, and these included 66 receptor-like protein kinase genes, three protein kinase genes, and one protein phosphatase gene. In particular, the serine/threonine kinase receptor (STKR) was similar to the Arabidopsis FRK1 induced by bacterial flagellins
, and a protein kinase was homologous to the Trichoderma-induced kinase (TIPK) of cucumber
. Furthermore, the STKR gene (JG391826) is induced by P. viticola in resistant grapevines
, suggesting that it plays a crucial role in the activation of specific defence processes against downy mildew.
Our data suggest that resistance induction was also mediated by hormone signalling and transcriptional reprogramming. Genes related to ET metabolism (two 1-aminocyclopropane-1-carboxylate oxidases) were induced by T39 treatment, together with two MYB (including MYB72), one FAMA, and one NAC transcription factors. Activation of ET metabolism was in agreement with previous data showing that JA/ET signals are involved in T39-induced resistance
[42, 58]. In Arabidopsis, MYB72 plays a crucial role in Trichoderma-induced resistance
 and is required in early signalling of rhizobacteria-induced resistance in Arabidopsis. The NAC gene is involved during oxidative stress
, and response to oxidative stress after T39 treatment was suggested by modulation of two peroxidases, three glutathione S-transferases (GSTs), and one thioredoxin. Moreover, defence-related genes (two STSs, a Chit, and a cellulose synthase) were also directly induced, indicating that the maximum expression level of these genes was probably reached after T39 treatment and that this was sufficient to contribute to defence against subsequent P. viticola inoculation. Other defence-related genes were pre-induced by T39 and further induced after P. viticola inoculation (cluster 2), indicating reinforcement of grapevine defence after pathogen inoculation.
Enhanced reaction of T39-treated plants to Plasmopara viticola
Plant resistance induced by beneficial microorganisms has been associated with faster and/or stronger activation of defence responses after pathogen inoculation
. P. vitiocla-responsive genes with enhanced expression in T39-treated plants were clustered to distinguish between those directly modulated (cluster 6) and those not modulated (cluster 7) by T39 treatment. These expression profiles provide strong support for the view that Trichoderma spp. may have a dual effect: it directly stimulates induction of some genes and further reinforces modulation of these and other genes after pathogen inoculation
. The dual effect was also reported for defence-gene modulation during resistance induced by sulfated laminarin
 and for phytoalexin accumulation during resistance induced by Rheum palmatum extracts
Enhanced reaction of T39-treated plants included induction of genes related to response to stimulus and response to stress categories, suggesting improvement of signalling pathways and activation of defence reactions in response to pathogen inoculation. In particular, 50 receptor-like kinase genes (35 in cluster 6 and 15 in cluster 7) and six protein kinase genes (one in cluster 6 and five in cluster 7) of signal transduction were primed in T39-treated plants. In agreement with our results, priming was associated with increased expression of mitogen-activated protein kinases (MPK3 and MPK6) in Arabidopsis plants
. Up-regulation of MPK genes was also associated with the reaction of resistant grapevines to P. viticola[4, 6], and post-translational modification may be the additional mechanism for activating MPKs in response to pathogens
. Moreover, expression of stress-related genes was enhanced in inoculated T39-treated plants, and these genes included PR
10, Chit3, and osmotin (OSM1). Similar profiles were observed for Arabidopsis PR genes during T. asperelloides T203 and Pseudomonas syringae interactions
, indicating common mechanisms of Trichoderma-induced resistance against biotrophs. Primed profiles were found in resistance-related genes (12 genes in cluster 6 and eight in cluster 7). Peronospora parasitica resistance genes (RPP) must be expressed at optimal levels to function against downy mildew in Arabidopsis, and specific profiles of grapevine RPP genes suggest fine tuning during T39-induced resistance.
A key role of transcriptional regulation and secondary metabolic processes in T39-induced resistance was also indicated by the priming profiles of four MYB, two WRKY, and 12 ET-responsive transcription factors (e.g., AP2), and of PAL and STSs genes. Moreover, the category DNA metabolic process was significantly overrepresented in ISR-primed genes. In particular, expression of the histone-lysine N-methyltransferases SUVR4 and ATX2 was enhanced after P. viticola inoculation, reflecting possible involvement of epigenetic modifications
[62–64] in T39-induced resistance.
Genes with enhanced expression in T39-treated plants have been previously related to defence against downy mildew in resistant grapevines. This was the case for PR genes (PR
10, and OSM1), invertase genes (INV1 and INV2), and genes related to secondary metabolism (PAL, STSs, STKR, copper-containing amine oxidase, polyphenol oxidase, and three resveratrol O-methyltransferases), for which up-regulation was greater in resistant than in susceptible grapevines upon P. viticola inoculation
[4, 8, 11–14]. The expression profiles of these marker genes suggest that the defence processes usually activated against downy mildew in resistant grapevines are partially stimulated in susceptible plants by T39-induced resistance.
Disease-related processes inhibited in T39-treated plants
Another important aspect of T39-induced resistance was evidenced by genes modulated by P. viticola exclusively in control plants (clusters 8 and 9). These genes were mainly down-regulated (57 and 55%, respectively), and they reflect exploitation of cellular resources and/or suppression of defence responses during the compatible interaction. Down-regulation caused by P. viticola in control plants involved categories of response to stress and primary metabolic processes. Many signalling components (kinase, phosphatase, calmodulin, and calcium signalling), transcription factors (WRKY and MYB), and disease resistance proteins were repressed, supporting the view that suppression of endogenous signals is required to establish the compatible interaction
. Three ABC transporters (e.g., ABC
C) were also repressed, and suppression of some ABC transporters increases the susceptibility to oomycete pathogens
. Specific alteration of carbohydrate metabolism by P. viticola in control plants was highlighted by modulation of glucosidase, galactosidase, mannosidase, and sucrose synthase (SS) genes. Moreover, repressed genes of cluster 9 were classified into the categories of energy metabolism (a phytochrome C, two malic enzymes, and a ribokinase) and defence response (five chitinases, three glucanases, three superoxide dismutases, and a callose synthase), reflecting disease-related process employed by P. viticola only in control plants. P. viticola might need to actively suppress plant defences during leaf colonisation through microbial effectors, as demonstrated in other oomycetes
[66, 67]. Particularly, hemibiotrophic and biotrophic species establish intimate associations with plants
[66, 67]. To establish infection, these pathogens must suppress the plant defence and manipulate the host metabolism by microbial effectors (virulence factors) that are translocated inside the plant cell or secreted into the extracellular space within plant tissue
[66, 68]. P. viticola effector genes have been recently described
, and modulation of host cell defences through virulence factors in susceptible grapevines has been indicated by histochemical
 and transcriptomic
 analyses. In agreement with the phenotypic observations, the specific modulation of grapevine genes in inoculated control but not in inoculated T39-treated plants indicates that T39-induced resistance acts by inhibiting some disease-related processes and probably by interfering with some pathogen-induced processes.
Specific transcriptional response of T39-treated plants to Plasmopara viticola
In contrast to broad down-regulation of genes in control plants (clusters 8 and 9), genes specifically modulated by P. viticola in T39-treated plants (cluster 10) were mainly induced (63%). These opposing reactions to P. viticola are particularly evident in genes related to protein metabolism, response to stimulus, and response to stress, which were mainly induced in T39-treated plants and mainly repressed in control plants. Up-regulation of genes associated with these categories has been observed in resistant grapevines
[4, 13, 71], indicating that the defence processes of resistant genotypes could be partially activated in susceptible varieties by T39-induced resistance. In particular, all NBS-encoding resistance (NBS
R) genes modulated by P. viticola in control plants (cluster 9) were repressed, whereas those with ISR-responsive specific profiles (cluster 10) or ISR-primed profiles (cluster 7) were mainly up-regulated. Opposing modulation of NBS
R genes probably reflects suppression of plant defence in control plants and activation of defence responses in T39-treated plants. Interestingly, the NBS
R genes of clusters 7 and 10 belong mainly to the Va component genome of grapevine
, indicating subgenome-dependent regulation of gene expression
 in grapevine.
Defence signals specifically activated in T39-treated plants included those mediated by auxin (two auxin transporters, two auxin-induced proteins, and two indole-3-acetic acid amido synthetases), ET (ACC oxidase and five ERF transcription factors), and JA (three lipoxygenases and two fatty acid desaturases). The role of JA/ET signalling pathways in T39-induced resistance has also been demonstrated by phytohormone-affected Arabidopsis mutants
 and by expression analysis of grapevine marker genes
. ISR is commonly regulated by JA/ET-dependent signalling pathways, and it is especially active against pathogens deterred by defences that are controlled by JA and ET
[33, 36]. The auxin response pathway is connected to the SA and JA/ET signalling networks
, and crosstalk between hormonal pathways
 enables the fine tuning of defence mechanisms so that the plant can tailor its response to the specific invader
. ET exerts its resistance-stimulating activity in concert with JA
, and JA pathways are involved in the reaction to P. viticola in resistant grapevines
[4, 6]. Thus, enhancement of JA/ET signals in T39-treated plants supports the view that increased resistance to downy mildew is mediated by partial activation of extant defence mechanisms normally activated in resistant genotypes. Reaction to the pathogen was also mediated by specific up-regulation of 59 receptor kinases, 10 protein kinases, two bHLHs genes, one MYB gene, and the NPR1.1 gene.
Our results also suggest that the cell redox balance is altered in T39-treated plants after pathogen inoculation. The reaction of T39-treated plants to pathogen inoculation included the induction four peroxidases and a GST. Antioxidant enzymes are often induced in response to pathogens, and alteration of oxidative-stress metabolism has a prominent role in the T39-induced resistance of grapevine to downy mildew
. Peroxidases play several important roles in pathogen resistance by contributing to the production of reactive oxygen species, the reinforcement of cell walls, and the production of phytoalexins. Accumulation of stilbene phytoalexin is one of the most important defence processes activated by resistant grapevines in response to P. viticola; genes of phenylpropanoid biosynthesis (flavanone 3-dioxygenase, laccase, and dihydroflavonol-4-reductase) were specifically induced in T39-treated plants, confirming activation of pathways known in resistant genotypes. However, additional defence mechanisms against P. viticola are activated in resistant genotypes. In particular, HR-related genes (Avr/Cf9, Hin1, and Hsr203j) were not induced and localised HR necrosis was not observed in T39-treated plants.
Common transcriptional response of control and T39-treated plants to Plasmopara viticola
Although specific transcriptional reprogramming of T39-treated plants was observed, 3,454 genes had comparable expression levels in control and T39-treated plants after P. viticola inoculation (clusters 4 and 5). The pathogen-responsive processes not affected by resistance induction were mainly related to primary metabolism and signal transduction. In particular, expression profiles of genes related to starch metabolism (up-regulation of α-amylase and sugar transporters, down-regulation of β-amylase, glucose-1-phosphate adenylyltransferases, sucrose phosphate synthase, and phosphoglucan water dikinase) indicated the source-to-sink transition of P. viticola-infected leaves
. Likewise, genes related to photosynthesis (two quinone oxidoreductases, a chlorophyllide oxygenase, a protochlorophyllide transporter, a chlorophyllase-2 and chlorophyll a-b binding proteins) and to the Calvin cycle (two rubisco genes and a phosphoglycerate kinase gene) were similarly modulated in inoculated control and T39-treated plants, possibly reflecting the establishment of a compatible interaction. P. viticola inoculation also resulted in the down-regulation of genes involved in the signal transduction processes (20 receptor-like protein kinase genes, 11 protein kinase genes, and five protein phosphatase genes) and defence response (23 probable disease resistance genes, six unspecified PR genes, a GST gene and a thaumatin-like gene
), indicating a pathogen-dependent suppression of the host reaction mechanisms.
Other markers of P. viticola infection in susceptible grapevines showed comparable modulation in T39-treated and control plants, such as enzymatic resistance protein
, zeaxanthin epoxidase
, isoforms of chalcone synthase
, and dihydroflavonol 4-reductase
. These results suggested that transcriptional changes associated with the compatible interaction
[5, 7, 10, 13] were not completely inhibited in T39-treated plants, which is consistent with the observation that downy mildew symptoms were reduced but not completely blocked by T39 treatment.